Prodrug of an ICE inhibitor

ABSTRACT

This invention describes an ICE inhibitor prodrug (I) having good bioavailability. 
     
       
         
         
             
             
         
       
     
     Compound I is useful for treating IL-1 mediated diseases such as rheumatoid arthritis, inflammatory bowel disease, Crohn&#39;s disease, ulcerative colitis, inflammatory peritonitis, septic shock, pancreatitis, traumatic brain injury, organ transplant rejection, osteoarthritis, asthma, psoriasis, Alzheimer&#39;s disease, myocardial infarction, congestive heart failure, Huntington&#39;s disease, atherosclerosis, atopic dermatitis, leukemias and related disorders, myelodysplastic syndrome, uveitis or multiple myeloma.

CROSS REFERENCE TO RELATED APPLICATIONS

Pursuant to Title 35, United States Code, §119 and §120, thisapplication is a divisional of U.S. patent application Ser. No.09/860,750, filed May 18, 2001, now U.S. Pat. No. 7,417,029, whichclaims benefit of U.S. Provisional Patent Application No. 60/205,439,filed May 19, 2000.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a novel interleukin-1β convertingenzyme (ICE) inhibitor in its prodrug form. The compound andpharmaceutical compositions thereof are useful as agents to treatinterleukin-1-(IL-1), apoptosis-, interferon-γ inducing factor- (IL-18),or interferon-γ (IFN-γ) mediated diseases, including inflammatorydiseases, autoimmune diseases, destructive bone disorders, proliferativedisorders, infectious diseases, and degenerative diseases. Thisinvention also relates to methods for inhibiting ICE activity anddecreasing IL-18 production and IFN-γ production and methods fortreating interleukin-1, apoptosis-, and interferon-γ-mediated diseasesusing the compositions of this invention.

BACKGROUND OF THE INVENTION

Interleukin-1 (IL-1) is a major pro-inflammatory and immunoregulatoryprotein that stimulates fibroblast differentiation and proliferation,the production of prostaglandins, collagenase and phospholipase bysynovial cells and chondrocytes, basophil and eosinophil degranulationand neutrophil activation. Oppenheim, J. H. et al, Immunology Today, 7,pp. 45-56 (1986). As such, it is involved in the pathogenesis of chronicand acute inflammatory and autoimmune diseases. For example, inrheumatoid arthritis, IL-1 is both a mediator of inflammatory symptomsand of the destruction of the cartilage proteoglycan in afflictedjoints. Wood, D. D. et al., Arthritis Rheum. 26, 975, (1983); Pettipher,E. J. et al., Proc. Natl. Acad. Sci. USA 71, 295 (1986); Arend, W. P.and Dayer, J. M., Arthritis Rheum. 38, 151 (1995). IL-1 is also a highlypotent bone resorption agent. Jandiski, J. J., J. Oral Path 17, 145(1988); Dewhirst, F. E. et al., J. Immunol. 8, 2562 1985). It isalternately referred to as “osteoclast activating factor” in destructivebone diseases such as osteoarthritis and multiple myeloma. Bataille, R.et al., Int. J. Clin. Lab. Res. 21(4), 283 (1992). In certainproliferative disorders, such as acute myelogenous leukemia and multiplemyeloma, IL-1 can promote tumor cell growth and adhesion. Bani, M. R.,J. Natl. Cancer Inst. 83, 123 (1991); Vidal-Vanaclocha, F., Cancer Res.54, 2667 (1994). In these disorders, IL-1 also stimulates production ofother cytokines such as IL-6, which can modulate tumor development(Tartour et al., Cancer Res. 54, p. 6243 (1994). IL-1 is predominantlyproduced by peripheral blood monocytes as part of the inflammatoryresponse and exists in two distinct agonist forms, IL-1α and IL-1β.Mosely, B. S. et al., Proc. Nat. Acad. Sci., 84, pp. 4572-4576 (1987);Lonnemann, G. et al., Eur. J. Immunol., 19, pp. 1531-1536 (1989).

IL-1β is synthesized as a biologically inactive precursor, pro-IL-1β.Pro-IL-1β lacks a conventional leader sequence and is not processed by asignal peptidase. March, C. J., Nature, 315, pp. 641-647 (1985).Instead, pro-IL-1β is cleaved by interleukin-1β converting enzyme (ICE)between Asp-116 and Ala-117 to produce the biologically activeC-terminal fragment found in human serum and synovial fluid. Sleath, P.R., et al., J. Biol. Chem., 265, pp. 14526-14528 (1992); A. D. Howard etal., J. Immunol., 147, pp. 2964-2969 (1991). ICE is a cysteine proteaselocalized primarily in monocytes. It converts precursor IL-1β to themature form. Black, R. A. et al., FEBS Lett., 247, pp. 386-390 (1989);Kostura, M. J. et al., Proc. Natl. Acad. Sci. U.S.A., 86, pp. 5227-5231(1989). Processing by ICE is also necessary for the transport of matureIL-1β through the cell membrane.

ICE (or caspase-1) is a member of a family of homologous enzymes calledcaspases. These homologs have sequence similarities in the active siteregions of the enzymes. Such homologs (caspases) include TX (orICE_(rel-II) or ICH-2) (caspase-4) (Faucheu, et al., EMBO J., 14, p.1914 (1995); Kamens J., et al., J. Biol. Chem., 270, p. 15250 (1995);Nicholson et al., J. Biol. Chem., 270 15870 (1995)), TY (orICE_(rel-III)) (caspase-5) (Nicholson et al., J. Biol. Chem., 270, p.15870 (1995); ICH-1 (or Nedd-2) (caspase-2) (Wang, L. et al., Cell, 78,p. 739 (1994)), MCH-2 (caspase-6), (Fernandes-Alnemri, T. et al., CancerRes., 55, p. 2737 (1995), CPP32 (or YAMA or apopain) (caspase-3)(Fernandes-Alnemri, T. et al., J. Biol. Chem., 269, p. 30761 (1994);Nicholson, D. W. et al., Nature, 376, p. 37 (1995)), CMH-1 (or MCH-3)(caspase-7) (Lippke, et al., J. Biol. Chem., 271(4), p 1825-1828(1996)); Fernandes-Alnemri, T. et al., Cancer Res., (1995)), Mch5(caspase-8) (Muzio, M. et. al., Cell 85(6), 817-827, (1996)), MCH-6(caspase-9) (Duan, H. et. al., J. Biol. Chem., 271(34), p. 16720-16724(1996)), Mch4 (caspase-10) (Vincenz, C. et. al., J. Biol. Chem., 272, p.6578-6583 (1997); Fernandes-Alnemri, T. et. al., Proc. Natl. Acad. Sci.93, p. 7464-7469 (1996)), Ich-3 (caspase-11) (Wang, S. et. al., J. Biol.Chem., 271, p. 20580-20587 (1996)), mCASP-12 (caspase-12), (Van deCraen, M. et. al., FEBS Lett. 403, p. 61-69 (1997); Yuan, Y. and Miura,M. PCT Publication WO95/00160 (1995)), ERICE (caspase-13), (Humke, E.W., et. al., J. Biol. Chem., 273(25) p. 15702-15707 (1998)), and MICE(caspase-14) (Hu, S. et. al., J. Biol. Chem., 273(45) p. 29648-29653(1998)).

Each of these ICE homologs, as well as ICE itself, is capable ofinducing apoptosis when overexpressed in transfected cell lines.Inhibition of one or more of these homologs with the peptidyl ICEinhibitor Tyr-Val-Ala-Asp-chloromethylketone results in inhibition ofapoptosis in primary cells or cell lines. Lazebnik et al., Nature, 371,p. 346 (1994).

Caspases also appear to be involved in the regulation of programmed celldeath or apoptosis. Yuan, J. et al., Cell, 75, pp. 641-652 (1993);Miura, M. et al., Cell, 75, pp. 653-660 (1993); Nett-Fiordalisi, M. A.et al., J. Cell Biochem., 17B, p. 117 (1993). In particular, ICE or ICEhomologs are thought to be associated with the regulation of apoptosisin neurodegenerative diseases, such as Alzheimer's and Parkinson'sdisease. Marx, J. and M. Baring a, Science, 259, pp. 760-762 (1993);Gagliardini, V. et al., Science, 263, pp. 826-828 (1994). Inhibition ofcaspases have also recently been shown to be effective in a murine modelof amylotropic lateral sclerosis. Li, M. et al.; Science, 288, pp.335-339 (2000). Therapeutic applications for inhibition of apoptosis mayinclude, among others, treatment of Alzheimer's disease, Parkinson'sdisease, stroke, myocardial infarction, spinal atrophy, and aging.

ICE has been demonstrated to mediate apoptosis (programmed cell death)in certain tissue types. Steller, H., Science, 267, p. 1445 (1995);Whyte, M. and Evan, G., Nature, 376, p. 17 (1995); Martin, S. J. andGreen, D. R., Cell, 82, p. 349 (1995); Alnemri, E. S., et al., J. Biol.Chem., 270, p. 4312 (1995); Yuan, J. Curr. Opin. Cell Biol., 7, p. 211(1995). A transgenic mouse with a disruption of the ICE gene isdeficient in Fasmediated apoptosis (Kuida, K. et al., Science 267, 2000(1995)). This activity of ICE is distinct from its role as theprocessing enzyme for pro-IL-1β. It is conceivable that in certaintissue types, inhibition of ICE may not affect secretion of matureIL-1β, but may inhibit apoptosis.

Enzymatically active ICE has been previously described as a heterodimercomposed of two subunits, p20 and p10 (20 kDa and 10 kDa molecularweight, respectively). These subunits are derived from a 45 kDaproenzyme (p45) by way of a p30 form, through an activation mechanismthat is autocatalytic. Thornberry, N. A. et al., Nature, 356, pp.768-774 (1992). The ICE proenzyme has been divided into severalfunctional domains: a prodomain (p14), a p22/20 subunit, a polypeptidelinker and a p10 subunit. Thornberry et al., supra; Casano et al.,Genomics, 20, pp. 474-481 (1994).

Full length p45 has been characterized by its cDNA and amino acidsequences. PCT patent applications WO 91/15577 and WO 94/00154. The p20and p10 cDNA and amino acid sequences are also known. Thornberry et al.,supra. Murine and rat ICE have also been sequenced and cloned. They havehigh amino acid and nucleic acid sequence homology to human ICE. Miller,D. K. et al., Ann. N.Y. Acad. Sci., 696, pp. 133-148 (1993); Molineaux,S. M. et al., Proc. Nat. Acad. Sci., 90, pp. 1809-1813 (1993). Thethree-dimensional structure of ICE has been determined at atomicresolution by X-ray crystallography. Wilson, K. P., et al., Nature, 370,pp. 270-275 (1994). The active enzyme exists as a tetramer of two p20and two p10 subunits.

Recently, ICE and other members of the ICE/CED-3 family have been linkedto the conversion of pro-IL-18 to IL-18 or to the production of IFN-γ invivo (PCT application PCT/US96/20843, publication no. WO 97/22619, whichis incorporated herein by reference). IL-18 is synthesized in vivo asthe precursor protein “pro-IL-18”.

Interleukin-18 (IL-18), formerly interferon-gamma inducing factor,(IGIF) is an approximately 18-kDa polypeptide that stimulates T-cellproduction of interferon-gamma (IFN-γ-). IL-18 is produced by activatedKupffer cells and macrophages in vivo and is exported out of such cellsupon endotoxin stimulation. Like IL-1β, IL-18 is synthesized as abiologically inactive precursor molecule lacking a single peptide, whichrequires cleavage into an active, mature molecule by IL-1β convertingenzyme. Dinerello, C. A. Methods, 19, pp 121-132 (1999). Thus, acompound that decreases IL-18 production would be useful as an inhibitorof such T-cell stimulation which in turn would reduce the levels ofIFN-γ production by those cells.

IFN-γ is a cytokine with immunomodulatory effects on a variety of immunecells. In particular, IFN-γ is involved in macrophage activation and Th1cell selection (F. Belardelli, APMIS, 103, p. 161 (1995)). IFN-γ exertsits effects in part by modulating the expression of genes through theSTAT and IRF pathways (C. Schindler and J. E. Darnell, Ann. Rev.Biochem., 64, p. 621 (1995); T. Taniguchi, J. Cancer Res. Clin. Oncol.,121, p. 516 (1995)).

Mice lacking IFN-γ or its receptor have multiple defects in immune cellfunction and are resistant to endotoxic shock (S. Huang et al., Science,259, p. 1742 (1993); D. Dalton et al., Science, 259, p. 1739 (1993); B.D. Car et al., J. Exp. Med., 179, p. 1437 (1994)). Along with IL-12,IL-18 appears to be a potent inducer of IFN-γ production by T cells (H.Okamura et al., Infection and Immunity, 63, p. 3966 (1995); H. Okamuraet al., Nature, 378, p. 88 (1995); S. Ushio et al., J. Immunol., 156, p.4274 (1996)).

IFN-γ has been shown to contribute to the pathology associated with avariety of inflammatory, infectious and autoimmune disorders anddiseases. Thus, compounds capable of decreasing IFN-γ production wouldbe useful to ameliorate the effects of IFN-γ related pathologies.

Accordingly, compositions and methods capable of regulating theconversion of pro-IL-18 to IL-18 would be useful for decreasing IL-18and IFN-γ production in vivo, and thus for ameliorating the detrimentaleffects of these proteins which contribute to human disorders anddiseases.

Caspase inhibitors represent a class of compounds useful for the controlof inflammation or apoptosis or both. Peptide and peptidyl inhibitors ofICE have been described (PCT patent applications WO 91/15577, WO93/05071, WO 93/09135, WO 93/12076, WO 93/14777, WO 93/16710, WO95/35308, WO 96/30395, WO 96/33209 and WO 98/01133; European patentapplications 503 561, 547 699, 618 223, 623 592, and 623 606; and U.S.Pat. Nos. 5,434,248, 5,710,153, 5,716,929, and 5,744,451). Such peptidylinhibitors of ICE have been observed to block the production of matureIL-1β in a mouse model of inflammation (vide infra) and to suppressgrowth of leukemia cells in vitro (Estrov et al., Blood, 84, 380a(1994)). However, due to their peptidic nature, such inhibitors aretypically characterized by undesirable pharmacologic properties, such aspoor cellular penetration and cellular activity, poor oral absorption,instability and rapid metabolism. Plattner, J. J. and D. W. Norbeck, inDrug Discovery Technologies, C. R. Clark and W. H. Moos, Eds. (EllisHorwood, Chichester, England, 1990), pp. 92-126. These properties havehampered their development into effective drugs.

Non-peptidyl compounds have also been reported to inhibit ICE in vitro.PCT patent application WO 95/26958; U.S. Pat. No. 5,552,400; Dolle etal., J. Med. Chem., 39, pp. 2438-2440 (1996). It is not clear howeverwhether these compounds have the appropriate pharmacological profiles tobe therapeutically useful.

WO 99/47545 describes a novel class of caspase inhibitors reported tohave a favorable in vivo profile. These inhibitors are represented bythe formula:

where X, Y, and R¹-R⁶ are various substituents. Among the many examplesof this class of inhibitors, the following structure was disclosed:

As is known in the art, the bioavailability of compounds within astructural class is difficult to predict. Relatively minor structuralmodifications often have a large impact on the absorption of a compound,its blood level concentrations and/or its half-life. For example, suchvariations in bioavailability can be seen from the data in WO 99/47545.As a consequence, structurally related compounds that have very good invitro potency may vary in therapeutic effectiveness.

Though progress has been made in improving the bioavailability of ICEinhibitors, there continues to be a need to identify and developcompounds that can effectively inhibit caspases, and that have improvedin vivo activity. Such compounds would be useful as agents forpreventing and treating chronic and acute forms of IL-1-, apoptosis-,IL-18-, or IFN-γ-mediated diseases, as well as inflammatory, autoimmune,destructive bone, proliferative, infectious, or degenerative diseases.

DESCRIPTION OF THE INVENTION

This invention provides a novel ICE inhibitor prodrug compound withsurprisingly good bioavailability in mammals. The compound isrepresented by formula I:

Compound I may be used alone or in combination with other therapeutic orprophylactic agents, such as antibiotics, immunomodulators or otheranti-inflammatory agents, for the treatment or prevention of diseasesmediated by IL-1, apoptosis, IL-18, or IFN-γ. This invention alsorelates to pharmaceutically acceptable derivatives and prodrugs of thecompound.

Compound I itself is a prodrug that undergoes bioconversion to an activeICE inhibitor II:

Compound I has better in vivo activity upon oral and/or intravenousadministration than the parent or active form of the drug. The activeform, aspartic aldehyde II, exhibits less than optimal in vivo activity,primarily because of poor bioavailability, and is therefore notwell-suited for direct therapeutic use. Generally, poor bioavailabilitymay result for any of the following reasons: the active form is notstable in the animal gut following ingestion, is not well-absorbedthrough the gut and/or is not well-delivered to the biologicalcompartment (e.g., the brain or lymphatic system) for which it isintended. While the prodrug I shows enhanced bioavailability relative toits active form II, this invention is not limited to any particularmechanism by which the bioavailability is enhanced.

Applicants studied a number of prodrug ICE inhibitors, includingexamples listed in the aforementioned WO 99/47545. Bioavailability wasdetermined by quantitating the amount of ICE inhibitor in rat plasmaafter oral administration, as described below. Compound I was found tohave unexpectedly improved bioavailability relative to other prodrug ICEinhibitors tested, including some that were closely related instructure.

The structure for compound I depicted herein is meant to include allstereochemical forms of the compound; i.e., the R and S configurationsfor each asymmetric center. Therefore, single stereochemical isomers aswell as enantiomeric and diastereomeric mixtures of the present compoundare within the scope of the invention. A preferred isomer is compoundI-A which has the “S” configuration at the carbon bearing the tert-butylgroup, has the “S” configuration at the 2-position of the proline ring,has the “S” configuration at the 3-position of the furanone ring, andhas the “R” configuration at the 2-ethoxy position of the furanone ring,as shown below:

Another preferred isomer is compound I-B:

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention.

The compounds of this invention may be prepared in general by methodsknown to those skilled in the art for analogous compounds, asillustrated by the general scheme below and by the preparative examplesbelow

Reagents

-   a)Cbz-Cl, NaHCO₃; b)H-Pro-OtBu, EDC, HOBT; c)10% Pd/C, H₂;    d)4-amino-3-chlorobenzoic acid, EDC, DIPEA; e)TFA; f)7, EDC, HOBT,    DIPEA; g)DMBA, Pd(PPh₃)₄

Certain of the intermediates that are useful for making compound I arebelieved to be novel. Accordingly, one embodiment of this inventionrelates to compounds represented by formula II:

wherein R is selected from hydrogen or an organic radical, preferablyhydrogen or a C₁₋₁₂ alkyl, and most preferably hydrogen or tert-butyl.It is understood that the organic radical moiety is a group that isunreactive toward the other functional groups in compound II. CompoundII is understood to include any of the four possible stereoisomers, aswell as mixtures thereof. A preferred isomer of II is represented byformula II-A:

wherein R is as described above.

Pharmaceutical compositions of this invention comprise a compound offormula I or a pharmaceutically acceptable salt thereof and apharmaceutically acceptable carrier. Such compositions may optionallycomprise an additional therapeutic agent. Such agents include, but arenot limited to, an anti-inflammatory agent, a matrix metalloproteaseinhibitor, a lipoxygenase inhibitor, a cytokine antagonist, animmunosuppressant, an anti-cancer agent, an anti-viral agent, acytokine, a growth factor, an immunomodulator, a prostaglandin or ananti-vascular hyperproliferation compound.

The term “pharmaceutically acceptable carrier” refers to a non-toxiccarrier that may be administered to a patient, together with a compoundof this invention, and which does not destroy the pharmacologicalactivity thereof.

Pharmaceutically acceptable carriers that may be used in thepharmaceutical compositions of this invention include, but are notlimited to, ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, woolfat and self-emulsifying drug delivery systems (SEDDS) such asα-tocopherol, polyethyleneglycol 1000 succinate, or other similarpolymeric delivery matrices.

In pharmaceutical composition comprising only a compound of formula I asthe active component, methods for administering these compositions mayadditionally comprise the step of administering to the subject anadditional agent. Such agents include, but are not limited to, ananti-inflammatory agent, a matrix metalloprotease inhibitor, alipoxygenase inhibitor, a cytokine antagonist, an immunosuppressant, ananti-cancer agent, an anti-viral agent, a cytokine, a growth factor, animmunomodulator, a prostaglandin or an anti-vascular hyperproliferationcompound.

The term “pharmaceutically effective amount” refers to an amounteffective in treating or ameliorating an IL-1-, apoptosis-, IL-18-, orIFN-γ-mediated disease in a patient. The term “prophylacticallyeffective amount” refers to an amount effective in preventing orsubstantially lessening IL-1-, apoptosis-, IL-18-, or IFN-γ-mediateddiseases in a patient.

The compounds of this invention may be employed in a conventional mannerfor controlling IL-18 and IFN-γ levels in vivo and for treating diseasesor reducing the advancement or severity of effects which are mediated byIL-1, apoptosis, IL-18, or IFN-γ. Such methods of treatment, theirdosage levels and requirements may be selected by those of ordinaryskill in the art from available methods and techniques.

For example, a compound of this invention may be combined with apharmaceutically acceptable adjuvant for administration to a patientsuffering from an IL-1-, apoptosis-, IL-18-, or IFN-γ-mediated diseasein a pharmaceutically acceptable manner and in an amount effective tolessen the severity of that disease.

Alternatively, the compounds of this invention may be used incompositions and methods for treating or protecting individuals againstIL-1, apoptosis-, IL-18, or IFN-γ-mediated diseases over extendedperiods of time. The compounds may be employed in such compositionseither alone or together with other compounds of this invention in amanner consistent with the conventional utilization of enzyme inhibitorsin pharmaceutical compositions. For example, a compound of thisinvention may be combined with pharmaceutically acceptable adjuvantsconventionally employed in vaccines and administered in prophylacticallyeffective amounts to protect individuals over an extended period of timeagainst IL-1-, apoptosis-, IL-18, or IFN-γ-mediated diseases.

The compounds of formula I may also be co-administered with othercaspase or ICE inhibitors to increase the effect of therapy orprophylaxis against various IL-1-, apoptosis-, IL-18-, or IFN-γ-mediateddiseases.

In addition, the compounds of this invention may be used in combinationwith either conventional anti-inflammatory agents or with matrixmetalloprotease inhibitors, lipoxygenase inhibitors and antagonists ofcytokines other than IL-1β.

The compounds of this invention can also be administered in combinationwith immunomodulators (e.g., bropirimine, anti-human alpha-interferonantibody, IL-2, GM-CSF, methionine enkephalin, interferon-alpha,diethyldithiocarbamate, tumor necrosis factor, naltrexone and EPO), withprostaglandins, or with antiviral agents (e.g., 3TC, polysulfatedpolysaccharides, ganiclovir, ribavirin, acyclovir, alpha interferon,trimethotrexate and fancyclovir) or prodrugs of these or relatedcompounds to prevent or combat IL-1-mediated disease symptoms such asinflammation.

When the compounds of this invention are administered in combinationtherapies with other agents, they may be administered sequentially orconcurrently to the patient. Alternatively, pharmaceutical orprophylactic compositions according to this invention comprise acombination of a compound of formula I and another therapeutic orprophylactic agent.

The pharmaceutical compositions of this invention may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. We prefer oraladministration. The pharmaceutical compositions of this invention maycontain any conventional non-toxic pharmaceutically-acceptable carriers,adjuvants or vehicles. In some cases, the pH of the formulation may beadjusted with pharmaceutically acceptable acids, bases or buffers toenhance the stability of the formulated compound or its delivery form.The term parenteral as used herein includes subcutaneous,intracutaneous, intravenous, intramuscular, intra-articular,intrasynovial, intrasternal, intrathecal, intralesional and intracranialinjection or infusion techniques.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, such as those described in Pharmacopeia Helvetica, or asimilar alcohol.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, and aqueous suspensions and solutions. Inthe case of tablets for oral use, carriers which are commonly usedinclude lactose and corn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oral administration in a capsuleform, useful diluents include lactose and dried corn starch. Whenaqueous suspensions and solutions and propylene glycol are administeredorally, the active ingredient is combined with emulsifying andsuspending agents. If desired, certain sweetening and/or flavoringand/or coloring agents may be added.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but are not limited to, cocoa butter, beeswax and polyethyleneglycols.

Topical administration of the pharmaceutical compositions of thisinvention is especially useful when the desired treatment involves areasor organs readily accessible by topical application. For applicationtopically to the skin, the pharmaceutical composition should beformulated with a suitable ointment containing the active componentssuspended or dissolved in a carrier. Carriers for topical administrationof the compounds of this invention include, but are not limited to,mineral oil, liquid petroleum, white petroleum, propylene glycol,polyoxyethylene polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active compound suspended ordissolved in a carrier. Suitable carriers include, but are not limitedto, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esterswax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Thepharmaceutical compositions of this invention may also be topicallyapplied to the lower intestinal tract by rectal suppository formulationor in a suitable enema formulation. Topically-administered transdermalpatches are also included in this invention.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

Dosage levels of between about 0.01 and about 100 mg/kg body weight perday, preferably between 0.5 and about 75 mg/kg body weight per day andmost preferably between about 1 and 50 mg/kg body weight per day of theactive ingredient compound are useful in a monotherapy for theprevention and treatment of IL-1-, apoptosis-, IL-18-, andIFN-γ-mediated diseases, including uveitis, inflammatory diseases,autoimmune diseases, destructive bone disorders, proliferativedisorders, infectious diseases, degenerative diseases, necroticdiseases, inflammatory peritonitis, osteoarthritis, acute pancreatitis,chronic pancreatitis, asthma, adult respiratory distress syndrome,glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus,scleroderma, chronic thyroiditis, Graves' disease, autoimmune gastritis,insulin-dependent diabetes mellitus (Type I), autoimmune hemolyticanemia, autoimmune neutropenia, thrombocytopenia, chronic activehepatitis, myasthenia gravis, inflammatory bowel disease, Crohn'sdisease, psoriasis, atopic dermatitis, graft vs. host disease,osteoporosis, multiple myeloma-related bone disorder, leukemias andrelated disorders, myelodysplastic syndrome, acute myelogenous leukemia,chronic myelogenous leukemia, metastatic melanoma, Kaposi's sarcoma,multiple myeloma, sepsis, septic shock, Shigellosis, Alzheimer'sdisease, Parkinson's disease, cerebral ischemia, myocardial ischemia,myocardial infarction, congestive heart failure, Huntington's disease,atherosclerosis, spinal muscular atrophy, multiple sclerosis,AIDS-related encephalitis, HIV-related encephalitis, aging, alopecia,neurological damage due to stroke, ulcerative collitis, infectioushepatitis, juvenile diabetes, lichenplanus, acute dermatomyositis,eczema, primary cirrhosis, uveitis, Behcet's disease, atopic skindisease, pure red cell aplasia, aplastic anemia, amyotrophic lateralsclerosis, nephrotic syndrome and systemic diseases or diseases witheffects localized in the liver or other organs having an inflammatory orapoptotic component caused by excess dietary alcohol intake or viruses,such as HBV, HCV, HGV, yellow fever virus, dengue fever virus, andJapanese encephalitis virus.

Typically, the pharmaceutical compositions of this invention will beadministered from about 1 to 5 times per day or alternatively, as acontinuous infusion. Such administration can be used as a chronic oracute therapy. The amount of active ingredient that may be combined withthe carrier materials to produce a single dosage form will varydepending upon the host treated and the particular mode ofadministration. A typical preparation will contain from about 5% toabout 95% active compound (w/w). Preferably, such preparations containfrom about 20% to about 80% active compound.

When the compositions of this invention comprise a combination of acompound of formula I and one or more additional therapeutic orprophylactic agents, both the compound and the additional agent shouldbe present at dosage levels of between about 10% to 80% of the dosagenormally administered in a monotherapy regime.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained. When thesymptoms have been alleviated to the desired level, treatment shouldcease. Patients may, however, require intermittent treatment on along-term basis upon any recurrence or disease symptoms.

As the skilled artisan will appreciate, lower or higher doses than thoserecited above may be required. Specific dosage and treatment regimensfor any particular patient will depend upon a variety of factors,including the activity of the specific compound employed, the age, bodyweight, general health status, sex, diet, time of administration, rateof excretion, drug combination, the severity and course of the disease,and the patient's disposition to the disease and the judgment of thetreating physician.

IL-1 or apoptosis mediated diseases which may be treated or prevented bythe compounds of this invention include, but are not limited to,inflammatory diseases, autoimmune diseases, proliferative disorders,infectious diseases, and degenerative diseases. The apoptosis-mediateddiseases which may be treated or prevented by the compounds of thisinvention include degenerative diseases.

IL-1 or apoptosis mediated inflammatory diseases which may be treated orprevented include, but are not limited to osteoarthritis, acutepancreatitis, chronic pancreatitis, asthma, and adult respiratorydistress syndrome. Preferably the inflammatory disease is osteoarthritisor acute pancreatitis.

IL-1 or apoptosis mediated autoimmune diseases which may be treated orprevented include, but are not limited to, glomerulonephritis,rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronicthyroiditis, Graves' disease, autoimmune gastritis, insulin-dependentdiabetes mellitus (Type I), autoimmune hemolytic anemia, autoimmuneneutropenia, thrombocytopenia, chronic active hepatitis, myastheniagravis, multiple sclerosis, inflammatory bowel disease, Crohn's disease,psoriasis, atopic dermatitis and graft vs. host disease. Preferably theautoimmune disease is rheumatoid arthritis, inflammatory bowel disease,Crohn's disease, psoriasis, or atopic dermatitis.

IL-1 or apoptosis mediated destructive bone disorders which may betreated or prevented include, but are not limited to, osteoporosis andmultiple myeloma-related bone disorder.

IL-1 or apoptosis mediated proliferative diseases which may be treatedor prevented include, but are not limited to, leukemias and relateddisorders, such as myelodysplastic syndrome, acute myelogenous leukemia,chronic myelogenous leukemia, metastatic melanoma, Kaposi's sarcoma, andmultiple myeloma.

IL-1 or apoptosis mediated infectious diseases which may be treated orprevented include, but are not limited to, sepsis, septic shock, andShigellosis.

IL-1 or apoptosis mediated degenerative or necrotic diseases which maybe treated or prevented by the compounds of this invention include, butare not limited to, Alzheimer's disease, Parkinson's disease, cerebralischemia, and myocardial ischemia. Preferably, the degenerative diseaseis Alzheimer's disease.

IL-1 or apoptosis-mediated degenerative diseases which may be treated orprevented by the compounds of this invention include, but are notlimited to, Alzheimer's disease, Parkinson's disease, cerebral ischemia,myocardial ischemia, spinal muscular atrophy, multiple sclerosis,AIDS-related encephalitis, HIV-related encephalitis, aging, alopecia,and neurological damage due to stroke.

Other diseases having an inflammatory or apoptotic component may betreated or prevented by the compounds of this invention. Such diseasesmay be systemic diseases or diseases with effects localized in the liveror other organs and may be caused by, for example, excess dietaryalcohol intake or viruses, such as HBV, HCV, HGV, yellow fever virus,dengue fever virus, and Japanese encephalitis virus.

IL-18- or IFN-γ-mediated diseases which may be treated or prevented bythe compounds of this invention include, but are not limited to,inflammatory, infectious, autoimmune, proliferative, neurodegenerativeand necrotic conditions.

IL-18- or IFN-γ-mediated inflammatory diseases which may be treated orprevented include, but are not limited to osteoarthritis, acutepancreatitis, chronic pancreatitis, asthma, rheumatoid arthritis,inflammatory bowel disease, Crohn's disease, ulcerative collitis,cerebral ischemia, myocardial ischemia and adult respiratory distresssyndrome. Preferably, the inflammatory disease is rheumatoid arthritis,ulcerative collitis, Crohn's disease, hepatitis or adult respiratorydistress syndrome.

IL-18- or IFN-γ-mediated infectious diseases which may be treated orprevented include, but are not limited to infectious hepatitis, sepsis,septic shock and Shigellosis.

IL-18- or IFN-γ-mediated autoimmune diseases which may be treated orprevented include, but are not limited to glomerulonephritis, systemiclupus erythematosus, scleroderma, chronic thyroiditis, Graves' disease,autoimmune gastritis, insulin-dependent diabetes mellitus (Type I),juvenile diabetes, autoimmune hemolytic anemia, autoimmune neutropenia,thrombocytopenia, myasthenia gravis, multiple sclerosis, psoriasis,lichenplanus, graft vs. host disease, acute dermatomyositis, eczema,primary cirrhosis, hepatitis, uveitis, Behcet's disease, atopic skindisease, pure red cell aplasia, aplastic anemia, amyotrophic lateralsclerosis and nephrotic syndrome. Preferably, the autoimmune disease isglomerulonephritis, insulin-dependent diabetes mellitus (Type I),juvenile diabetes, psoriasis, graft vs. host disease or hepatitis.

More preferred diseases or conditions which may be treated or preventedinclude rheumatoid arthritis, inflammatory bowel disease, includingCrohn's disease and ulcerative colitis, inflammatory peritonitis,amyotrophic lateral sclerosis, septic shock, pancreatitis, traumaticbrain injury, organ transplant rejection, osteoporosis, osteoarthritis,asthma, uveitis, psoriasis, Alzeheimer's disease, myocardial infarction,congestive heart failure, Huntington's disease, atherosclerosis, atopicdermatitis, or leukemias and related disorders, such as myelodysplasticsyndrome or multiple myeloma.

Accordingly, one embodiment of this invention provides a method fortreating or preventing an IL-1 or apoptosis mediated disease in asubject comprising the step of administering to the subject anycompound, pharmaceutical composition, or combination described hereinand a pharmaceutically acceptable carrier.

Another embodiment of this invention provides a method for decreasingIL-18 production in a subject comprising the step of administering tothe subject any compound, pharmaceutical composition, or combinationdescribed herein and a pharmaceutically acceptable carrier.

Yet another embodiment of this invention provides a method fordecreasing IFN-γ production in a subject comprising the step ofadministering to the subject any compound, pharmaceutical composition,or combination described herein and a pharmaceutically acceptablecarrier.

Although this invention focuses on the use of the compounds disclosedherein for preventing and treating IL-1, apoptosis-, IL-18, andIFN-□-mediated diseases, the compounds of this invention can also beused as inhibitory agents for other cysteine proteases.

The compounds of this invention are also useful as commercial reagentswhich effectively bind to caspases or other cysteine proteasesincluding, but not limited to ICE. As commercial reagents, the compoundsof this invention, and their derivatives, may be used to blockproteolysis of a target peptide in biochemical or cellular assays forICE and ICE homologs or may be derivatized to bind to a stable resin asa tethered substrate for affinity chromatography applications. These andother uses which characterize commercial cysteine protease inhibitorswill be evident to those of ordinary skill in the art.

In order that this invention be more fully understood, the followingexamples are set forth. These examples are for the purpose ofillustration only and are not to be construed as limiting the scope ofthe invention in any way.

SYNTHETIC EXAMPLES Preparation of1-[2-(4-amino-3-chloro-benzoylamino)-3,3-dimethyl-butyryl]-pyrrolidine-2-carboxylicacid (2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide (I-A)2-Benzyloxycarbonylamino-3,3-dimethyl-butyric acid (2)

To a solution of L-tert-leucine (1) (50.0 g, 38.0 mmol) and NaHCO₃ (96.0g, 114 mmol) in ice (500 g) and water (500 ml) was added benzylchloroformate (65.0 ml, 74.0 mmol) and the reaction stirred at 0° C. for3 hours then at room temperature for 18 hours. 0.1N Na₂CO₃ was addeduntil the oily layer dissolved and the solution was washed with 10%EtOAc in hexanes (2×500 ml). The iced aqueous phase was acidified to pH1 using 12N HCl then extracted using EtOAc (3×350 ml). The combinedorganic extracts were dried over Na₂SO₄, filtered and evaporated to givethe title compound as a colorless oil (82.4 g, 81.5% yield):

¹H-NMR (500 MHz, CDCl₃) δ1.02 (s, 9H), 4.22 (d, 1H), 5.10-5.14 (m, 2H),5.31 (d, 1H), 7.26-7.37 (m, 5H).

1-(2-Benzyloxycarbonylamino-3,3-dimethyl-butyryl)-pyrrolidine-2-carboxylicacid tert-butyl ester (3)

To a solution of 2 (6.01 g, 2.0 mmol) in CH₂Cl₂ (30 ml) and anhydrousDMF (dimethylformamide)(10 ml) at 0° C. was added HOBT (3.16 g, 2.0mmol), EDC (1-(3-dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride)(7.19 g, 4.0 mmol) and L-proline-tert-butyl ester (4.22 g, 2.0 mmol).The solution was stirred at 0° C. for 10 minutes, then at roomtemperature for 5 hours. The solvents were evaporated in-vacuo and theresulting oil dissolved in EtOAc which was washed with H₂O (3×200 ml)and brine (200 ml). The organic phase was dried over anhydrous Na₂SO₄,filtered and evaporated to give the crude product. Flash chromatographyon silica gel using hexanes/EtOAc (95/5 to 80/20%) afforded the titlecompound as a colorless oil (8.30 g, 87.5% yield): ¹H-NMR (500 MHz,CDCl₃) □ 1.04 (s, 9H), 1.45 (s, 9H), 1.89-1.96 (m, 2H), 2.02-2.05 (m,1H), 2.18-2.22 (m, 1H), 3.65-3.69 (m, 1H), 3.79-3.82 (m, 1H), 4.34-4.37(m, 2H), 5.03-5.19 (m, 2H), 5.53 (d, 1H), 7.26-7.38 (5H).

Synthesis of1-[2-(4-amino-3-chloro-benzoylamino)-3,3-dimethyl-butyryl]-pyrrolidine-2-carboxylicacid tert-butyl ester (4)

To a solution of 3 (19.0 g, 45.4 mmol) in MeOH (200 mL) was added 10%activated Pd on C (2.0 g) in EtOAc (50 mL) and the reaction stirredunder H₂ for 18 hours. The solution was filtered through Celite and thesolvent evaporated to yield a viscous, colorless oil. The free amine wasdissolved in dry CH₂Cl₂/DMF (2:1, 120 mL), the solution cooled to 0° C.and 4-amino-3-chlorobenzoic acid (7.79 g, 45.4 mmol) and DIPEA (7.90 mL,45.4 mmol) were added. The reaction was stirred for 10 minutes, then EDC(11.32 g, 59.1 mmol) was added. The mixture was stirred at 0° C. for 30minutes then at room temperature for 18 hours. The solution was dilutedwith EtOAc (300 mL), washed with 0.5N NaHSO₄ (2×250 mL), 10% NaHCO₃(2×250 mL), saturated NaCl (150 mL), dried over MgSO₄, and evaporated todryness. Flash column chromatography on silica gel using CH₂Cl₂/MeOH,(99/1 to 98/2%) yielded the title compound as a white solid (19.25 g,97% yield): ¹H-NMR (500 MHz, CDCl₃) δ 1.12 (s, 9H), 1.48 (s, 9H),1.85-1.99 (m, 2H), 2.01-2.13 (m, 1H), 2.18-2.29 (m, 1H), 3.63-3.73 (m,1H), 3.84-3.93 (m, 1H), 4.30-4.41 (m, 1H), 4.86 (d, 1H), 6.73 (d, 1H),7.51 (d, 1H), 7.73 (s, 1H). Analytical HPLC (cyano column): 12.59 min.LC-MS (ES+) m/e=438.5 (M+H).

Synthesis of1-[2-(4-amino-3-chloro-benzoylamino)-3,3-dimethyl-butyryl]-pyrrolidine-2-carboxylicacid (5)

To a solution of 4 (15.9 g, 36.3 mmol) in CH₂Cl₂ (30 mL) was added TFA(trifluoroacetic acetic acid) (30 mL) and the solution stirred at roomtemperature for 3 hours under N₂. The reaction was transferred to abeaker (1 L) and diluted with CH₂Cl₂ (60 mL). To the solution at 0° C.was added solid NaHCO₃ (39 g, 46 mmol) and stirred for 15 minutes beforepartitioning between EtOAc (300 mL) and H₂O (300 mL). After extractionthe aqueous layer was acidified to pH 4-5 and extracted with EtOAc. Theorganic layer was dried (MgSO₄) and evaporated to dryness to give 5 as awhite solid (14.0 g, quantitative yield):

¹H-NMR (500 MHz, CDCl₃) δ1.08 (s, 9H), 1.97-2.22 (m, 3H), 2.29-2.41 (m,1H), 3.71-3.78 (m, 1H), 3.90-3.98 (m, 1H), 4.55-4.62 (m, 1H), 4.86 (d,1H), 6.64 (d, 1H), 6.74 (d, 1H), 7.53 (d, 1H), 7.74 (s, 1H). AnalyticalHPLC (cyano column): 8.24 min. LC-MS (ES+) m/e=382.4 (M+H).

Synthesis of1-[2-(4-amino-3-chloro-benzoylamino)-3,3-dimethyl-butyryl]-pyrrolidine-2-carboxylicacid (2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide (1-A)

To a solution of 6 (5.05 g, 22.0 mmol) in CH₂Cl₂ (50 mL) at 0° C. wasadded 1,3-dimethylbarbituric acid (DMBA) (3.78 g, 24.2 mmol) andPd(PPh₃)₄ (0.15 g, 0.13 mmol). After 10 minutes, a solution of 5 (8.40g, 22.0 mmol) in DMF (25 mL) was added followed by diisopropylethylamine(DIPEA) (7.66 mL, 44.1 mmol), (2.98 g, 22.0 mmol) and EDC (5.06 g, 26.4mmol). The solution was stirred at 0° C. for 10 minutes then at roomtemperature for 18 hours. The reaction was diluted with EtOAc (200 mL),washed with 0.5N NaHSO₄ (2×200 mL), 10% NaHCO₃ (2×200 mL), saturatedNaCl (1×150 mL), dried over anhydrous MgSO₄, and evaporated to dryness.Flash column chromatography on silica gel using CH₂Cl₂/MeOH, (99/1 to98/2%) afforded the title compound as a white solid (11.20 g, 77%yield): ¹H-NMR (500 MHz, CDCl₃) δ 1.08 (s, 9H), 1.27 (t, 3H), 1.85-1.99(m, 1H), 2.00-2.06 (m. 1H), 2.07-2.18 (m, 1H), 2.32-2.48 (m, 2H),2.78-2.89 (m, 1H), 3.62-3.76 (m, 2H), 3.82-3.96 (m, 2H), 4.39 (s, 1H),4.54-4.60 (m, 1H), 4.62-4.76 (m, 1H), 4.85 (d, 1H), 6.57 (d, 1H), 6.73(d, 1H), 7.38 (d, 1H), 7.49 (d, 1H), 7.72 (s, 1H). Analytical HPLC(cyano column): 13.10 min. LC-MS (ES⁺) m/e=509.4 (M+H), m.p.=96-99° C.

Oral Pharmacokinetic Studies

Male Sprague-Dawley rats (Harlan, Indianapolis, Ind., 300-350 g) wereanesthetized by an intramuscular injection of ketamine/rompun mixture. APE-50 cannula was inserted in the right carotid artery for arterialblood sampling. The rats were allowed to recover from surgery overnight(16 hours) prior to being used in the study. Test compounds wereadministered orally at 50 mg/kg 100% propylene glycol (PG) at a dosevolume of 10 mL/kg. Blood samples (˜0.30 mL) were removed at 0.25, 0.50,1.0, 1.5, 2, 3, 4, 6, and 8 hours post-dose, plasma separated bycentrifugation and stored at −80° C. pending analysis. Quantification ofthe plasma samples was conducted using a gradient HPLC/MS/MS similar tothe one detailed below:

HPLC/MS/MS Method for the quantitation of ICE inhibitors in rat plasma

Sample Preparation

1. 100 μl of plasma are aliquotted into Ependorf centrifuge vials.

2. An equal volume of acetonitrile is added to the plasma to precipitateplasma proteins.

3. Samples are vortexed for 2 minutes, and centrifuged at 14,000 rpmsfor 3 minutes.

4. 100 μl of the supernatant is loaded into 12 mm HPLC liquid samplervials.

5. A 20 μl addition of external standard is added to the 100 μl aliquotto monitor variation in instrumental response.

6. 10 μl of sample is injected for analysis via the mass spectrometer.

HPLC Instrumental Parameters

HPLC: Hewlett Packard HP1100 Binary Solvent Delivery System.

HPLC Gradient Conditions

-   A=H₂O 0.2% Formic Acid-   B=Acetonitrile 0.2% Formic Acid    Mobile Phase

Time (min) % A % B 0 100 0 2 100 0 5 0 100 11 0 100 11.5 100 0 15 100 0HPLC Analytical Column: Keystone Phenyl-1 Hypersil 2.0×100 mm, 5μ 120 Åpore size, P/N# 105-36-2

Injection Volume: 10 μl Flow Rate: 0.20 mL/min.Mass Spectrometry Instrumental Parameters

Instrument: Micromass Quattro Ultima, Tandem Mass SpectrometerIonization Technique: Orthogonal spray (ESI) Polarity: Positive DwellTime: 300 msec Pause Time: 5 msec Scan time: 0.9 sec Scan Mode: MRM(Multiple Reaction Monitoring) Ions/Transitions: For compound I-A m/z509.1-243.1 For compound II m/z 481.1-215.1Pharmacokinetic Parameters

Pharmacokinetic analysis of these plasma concentration data wasconducted using noncompartmental methods. The area under the curve(AUC_((0-t))) was estimated from time zero to the last measured timepoint using the linear trapezoidal rule. The rate of elimination (ke)was estimated by log-linear regression from the terminal phase of theplasma concentration-time curves. Area under the tail of the curve wasestimated as the ratio of the last measured concentration to ke. Thearea under the curve from time zero to infinity (AUC(0-∞)) was obtainedby addition of the area under the tail to AUC(0-t). Eliminationhalf-life was estimated as 0.693/ke. The observed values for the peakplasma concentration (Cmax) were recorded.

TABLE 1 Oral Pharmacokinetic Data Cmax AUC t 1/2 Example (μg/mL)(μgXh/mL) (hrs)

1.8 2.18 2.9

0.51 1.35 0.25

4.27 11.7 2.5

Table 1 above compares the pharmacokietic values of compound I withcompounds A and B that are closely related in structure. As can be seenfrom the data, Cmax and AUC are much higher for compound I than for theother two compounds.

While we have described a number of embodiments of this invention, it isapparent that our basic constructions may be altered to provide otherembodiments that utilize the products and processes of this invention.

1. A method for treating a disease selected from osteoarthritis,pancreatitis, rheumatoid arthritis, insulin-dependent diabetes mellitus(Type I), inflammatory bowel disease, Crohn's disease, psoriasis, graftvs host disease, osteoporosis, amyotrophic lateral sclerosis (ALS),sepsis, septic shock, cerebral ischemia, myocardial ischemia,Huntington's disease, multiple sclerosis, neurological damage due tostroke, ulcerative colitis, traumatic brain injury, and organ transplantrejection, in a patient having one or more of the aforementioneddiseases comprising the step of administering to said patient apharmaceutical composition comprising a compound of formula I;

and a pharmaceutically acceptable carrier, adjuvant or vehicle.
 2. Themethod according to claim 1, wherein the disease is rheumatoidarthritis, inflammatory bowel disease, Crohn's disease, ulcerativecolitis, amyotrophic lateral sclerosis, septic shock, pancreatitis,traumatic brain injury, organ transplant rejection, osteoporosis,osteoarthritis, psoriasis, myocardial infarction, or Huntington'sdisease.
 3. The method according to claim 2, wherein the disease isinflammatory bowel disease, Crohn's disease, or ulcerative colitis.